The steroid hormone progesterone is a potent hormonal effector implicated in the control of proliferation, differentiation and development of mammary and uterine tissues. Clark, C. L. and R. L. Sutherland, Progestin Regulation of Cellular Proliferation, 11 Endocrine Rev., 266-301 (1990). The endocrine effects of this hormone are manifest only in cells containing a specific intracellular receptor, the progesterone receptor (PR). The interaction between PR and its cognate ligand (progesterone) induces a series of structural and functional changes in the protein, leading ultimately to an association of the receptor with specific DNA sequences in the regulatory regions of target genes. The cellular and promoter context of the bound receptor determines the phenotypic consequence of this interaction. O'Malley, B. W., The Steroid Receptor Superfamily: More Excitement Predicted for the Future, 4 Mol. Endocrinol., 363-369 (1990); Beato, M., Gene Regulation by Steroid Hormones, 56 Cell 335-344 (1989).
The progesterone receptor (PR) is a member of a closely related sub-group of intracellular receptors that includes the androgen, mineralocorticoid, glucocorticoid and estrogen receptors. Evans, R. M., The Steroid and Thyroid Receptor Superfamily, 240 Science, 889-895 (1988). Within this sub-group, human PR is unique in that it occurs in target tissues as two distinct subtypes, PR-A and PR-B, of 94 and 114 kDa, respectively. Schrader, W. T. and B. W. O'Malley, Progesterone-Binding Components of Chick Oviduct: Characterization of Purified Subunits, 247 J. Biol. Chem., 51-59 ( 1972); Horwitz, K. B. and P. S. Alexander, In situ Photolinked Nuclear Progesterone Receptors of Human Breast Cancer Cells: Subunit Molecular Weights After Transformation and Translocation, 113 Endocrinology, 2195-2201 (1983). The PR-B isoform contains an N-terminal fragment of 164 amine acids (B 164) which is absent on the PR-A isoform. It is likely that both forms can arise as a result of either alternate initiation of translation from the same mRNA or by transcription from alternate promoters within the same gene. Conneely, O. M., Maxwell, B. L., Toft, D. O., Schrader, W. T. and B. W. O'Malley, The A and B Forms of the Chicken Progesterone Receptor Arise by Alternate Initiation of Translation of a Unique mRNA, 149 Biochem. Bioph. Res. Comm., 493-501 (1987); Kastner, P., Krust, A., Turcotte, B., Stropp, U., Tora, L., Gronemeyer, H. and P. Chambon, Two Distinct Estrogen-Regulated Promoters Generate Transcripts Encoding the Two Functionally Different Human Progesterone Receptor Forms A and B, 9 EMBO J., 1603-1614 (1990). Interestingly, Kastner et al., have identified two distinct promoters in the hPR gene. These promoters which regulate the synthesis of specific transcripts corresponding to hPR-A and hPR-B are regulated independently. Id. It is likely therefore, that the expression levels of PR-A and PR-B can differ with respect to each other in certain target tissues.
The biochemical properties of the PR isoforms have been analyzed in vitro, where they displayed similar DNA and hormone binding affinities. Lessey, B. A., Alexander, P. S. and K. B. Horwitz, The Subunit Structure of Human Breast Cancer Progesterone Receptors: Characterization by Chromatography and Photoaffinity Labeling, 112 Endocrinology, 1267-1283 (1983); Christensen, K., Estes, P. A., Onate, S. A., Beck, C. A., DeMarzo, A., Altmann, M., Lieberman, B. A., St. John, J., Nordeen, S. K. and D. P. Edwards, Characterization and Functional Properties of the A and B Forms of Human Progesterone Receptors Synthesized in a baculovirus System, 5 Mol. Endocrinol., 1755-1770 (1991). However, when analyzed in reconstituted progesterone response systems in heterologous cells it became apparent that hPR-A and hPR-B have different promoter specificities. Kastner et al.; Meyer, M. E., Pornon, A., Ji, J., Bocquel, M. T., Chambon, P. and H. Gronemeyer, Agonist and Antagonist Activities of RU486 on the Functions of the Human Progesterone Receptor, 9 EMBO J., 3923-3932 (1990). A similar result was obtained when the transcriptional activities of chicken PR-A and PR-B were assessed. Tora, L., Gronemeyer, H., Turcotte, B., Gaub, M. P. and P. Chambon, The N-terminal Region of the Chicken Progesterone Receptor Specifies Target Gene Activation, 333 Nature, 185-188 (1988); Bocquel, M. T., Kumar, V., Stricker, C., Chambon, P. and H. Gronemeyer, The Contribution of the N- and C-terminal Regions of Steroid Receptors to Activation of Transcription is both, Receptor and Cell-specific, 17 Nucleic Acids Res., 2581-2595 (1989).
Two distinct regions within hPR required for transcriptional activation (TAFs) have been identified, TAF1 located in the amino terminus and TAF2 within the carboxyl terminus. Interestingly, both TAFs are contained within PR-A and PR-B. The B164 region, unique to hPR-B, does not contain additional transcriptional activators but is required for maximal TAF1 function in the context of the full-length receptor. Meyer, M. E., Qurin-Stricker, C., Lerouge, T., Bocquel, M. T. and H. Gronemeyer, A Limiting Factor Mediates the Differential Activation of Promoters by the Human Progesterone Receptor Isoforms, 267 J. Biol. Chem., 10882-10887 (1992). It is possible, therefore, that in cell and promoter contexts where TAF1 activity is required, that PR-B will be a more efficient transcriptional regulator than PR-A.
The precise mechanism by which the promoter bound receptor exerts its transcriptional effect is unclear at present. The reconstitution of steroid receptor dependent transcription in vitro has been informative in this regard. Klein-Hitpass, L., Tsai, S. Y., Weigel, N. L., Allan, G. A., Riley, D., Rodriguez, R., Schrader, W. T., Tsai, M. J. and B. W. O'Malley, The Progesterone Receptor Stimulates Cell-free Transcription by Enhancing the Formation of a Stable Pre-initiation Colnflex, 60 Cell, 247-257 (1990). On chromatin free templates it is clear that at least one of the functions of the receptor is to recruit and/or stabilize the transcription pre-initiation complex at the core promoter. It is probable, however, that in the context of the intact cell, additional factors and processes in unison with the activated receptor are required for appropriate function. The existence of "adapters" or "co-factors" which influence the interaction between bound receptor and the general transcription apparatus has been implicated by several studies. Shemshedin, L., Ji, J., Brou, C., Chambon, P. and H. Gronemeyer, In vitro Activity of the Transcription Activation Functions of the Progesterone Receptor, 267 J. Biol. Chem., 1834-1839 (1992); Tora et al. It is likely that these "co-factors" are differentially expressed in cells and in the case of the progesterone receptor may be the determinants of the cellular and promoter context preferences of the PR-A and PR-B.
General assays and methods for detecting the transcriptional activity of an intracellular receptor (IR) when exposed to a known ligand or unknown compound have been developed. For example, U.S. Pat. No. 5,071,773, describes an assay by which hormone IRs, ligands for these receptors, and proteins having transcriptional activating properties of a hormone IR can be identified. Generally, the assay involves use of a cell which contains both DNA encoding a hormone response element (i.e., a promoter) linked to an operative reporter gene and DNA encoding an IR protein. When a suitable hormone or ligand is exposed to the cell, a hormone-IR complex forms and is delivered to an appropriate DNA binding region, thereby activating the hormone response element, which in turn leads to expression of the product encoded by the reporter gene. Thereafter, activation of the reporter gene is detected by standard procedures used for detecting the product of a reporter gene, as an indication of the relative transcriptional activity of the hormone or ligand on the cellular system.